Method for the detection of cytosine methylations in immobilized DNA samples

ABSTRACT

A method is described for the analysis of cytosine methylation patterns in genomic DNA samples. In the first method step, the genomic DNA is isolated from cells or other accompanying materials and bound essentially irreversibly to a surface. Then the DNA bound to the surface is treated, preferably with a bisulfite, in such a way that cytosine is converted into a base that is different in its base pairing behavior in the DNA duplex, while 5-methylcytosine remains unchanged. Then the reagents that were used are removed in a washing step. Finally, selected segments of the immobilized DNA are amplified in a polymerase reaction and the amplified products are investigated with respect to their sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/981,357, filed Oct. 31, 2007, which in turn is acontinuation of U.S. patent application Ser. No. 10/416,624, filed Jan.5, 2004, now U.S. Pat. No. 7,407,749, both of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention concerns a method for the detection of cytosinemethylation in DNA samples.

The levels of observation that have been well studied in molecularbiology according to developments in methods in recent years include thegenes themselves, the transcription of these genes into RNA and thetranslation to proteins therefrom. During the course of development ofan individual, which gene is turned on and how the activation andinhibition of certain genes in certain cells and tissues are controlledcan be correlated with the extent and nature of the methylation of thegenes or of the genome. In this regard, pathogenic states are alsoexpressed by a modified methylation pattern of individual genes or ofthe genome.

5-Methylcytosine is the most frequent covalently modified base in theDNA of eukaryotic cells. For example, it plays a role in the regulationof transcription, in genetic imprinting and in tumorigenesis. Theidentification of 5-methylcytosine as a component of genetic informationis thus of considerable interest. 5-Methylcytosine positions, however,cannot be identified by sequencing, since 5-methylcytosine has the samebase-pairing behavior as cytosine. In addition, in the case of a PCRamplification, the epigenetic information which is borne by the5-methylcytosines is completely lost.

A relatively new method that in the meantime has become the most widelyused method for investigating DNA for 5-methylcytosine is based on thespecific reaction of bisulfite with cytosine, which, after subsequentalkaline hydrolysis, is then converted to uracil, which corresponds inits base-pairing behavior to thymidine. In contrast, 5-methylcytosine isnot modified under these conditions. Thus, the original DNA is convertedso that methylcytosine, which originally cannot be distinguished fromcytosine by its hybridization behavior, can now be detected by“standard” molecular biology techniques as the only remaining cytosine,for example, by amplification and hybridization or sequencing. All ofthese techniques are based on base pairing, which is now fully utilized.The prior art, which concerns sensitivity, is defined by a method thatincorporates the DNA to be investigated in an agarose matrix, so thatthe diffusion and renaturation of the DNA is prevented (bisulfite reactsonly on single-stranded DNA) and all precipitation and purificationsteps are replaced by rapid dialysis. (Olek A, Oswald J, Walter J. Amodified and improved method for bisulphite based cytosine methylationanalysis. Nucleic Acids Res. 1996 Dec. 15; 24(24):5064-6). Individualcells can be investigated by this method, which illustrates thepotential of the method. Of course, up until now, only individualregions of up to approximately 3000 base pairs long have beeninvestigated; a global investigation of cells for thousands of possiblemethylation analyses is not possible. Of course, this method also cannotreliably analyze very small fragments of small quantities of sample.These are lost despite the protection from diffusion through the matrix.

An overview of other known possibilities for detecting 5-methylcytosinescan be derived from the following review article: Rein T, DePamphilis ML, Zorbas H. Identifying 5-methylcytosine and related modifications inDNA genomes. Nucleic Acids Res. 1998 May 15; 26(10):2255-64.

The bisulfite technique has been previously applied only in research,with a few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Dörfler W,Horsthemke B. A single-tube PCR test for the diagnosis of Angelman andPrader-Willi syndrome based an allelic methylation differences at theSNRPN locus. Eur J Hum Genet. 1997 March-April; 5(2):94-8). However,short, specific segments of a known gene have always been amplifiedafter a bisulfite treatment and either completely sequenced (Olek A,Walter J. The pre-implantation ontogeny of the H19 methylation imprint.Nat. Genet. 1997 November; 17(3):275-6) or individual cytosine positionshave been detected by a “primer extension reaction” (Gonzalgo M L, JonesPa. Rapid quantitation of methylation differences at specific sitesusing methylation-sensitive single nucleotide primer extension(Ms-SNuPE) Nucleic Acids Res. 1997 Jun. 15; 25(12):2529-31, WO Patent95-00669) or an enzyme cleavage (Xiong Z, Laird P W COBRA: a sensitiveand quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15;25(12):2532-4). Detection by hybridization has also been described (Oleket al., WO-A 99-28498).

Urea improves the efficiency of bisulfite treatment prior to sequencingof 5-methylcytosine in genomic DNA (Paulin R, Grigg G W, Davey M W,Piper A A. Urea improves efficiency of bisulphite-mediated sequencing of5′-methylcytosine in genomic DNA. Nucleic Acids Res. 1998 Nov. 1;26(21):5009-10).

Other publications which are concerned with the application of thebisulfite technique for the detection of methylation in the case ofindividual genes are: Grigg G, Clark S. Sequencing 5-methylcytosineresidues in genomic DNA. Bioassays. 1994 June; 16(6):431-6, 431;Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Dörfler W.Imprinted segments in the human genome: different DNA methylationpatterns in the Prader-Willi/Angelman syndrome region as determined bythe genomic sequencing method. Hum Mol Genet. 1997 March; 6(3):387-95;Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis onindividual chromosomes: improved protocol for bisulphite genomicsequencing. Nucleic Acids Res. 1994 Feb. 25; 22(4):695-6; Martin V,Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicatesa correlation between DNA hypomethylation in the 5′ region of the pS2gene and in its expression in human breast cancer cell lines. Gene. 1995May 19; 157(1-2):261-4; WO 97/46705, WO 95/15373 and WO 95/45560.

Another known method is so-called methylation-sensitive PCR (Herman J G,Graff J R, Myohanen S, Nelkin B D, Baylin S B (1996),Methylation-specific PCR: a novel PCR assay for methylation status ofCpG islands. Proc Natl Acad Sci U S A. September 3; 93(18):9821-6). Forthis method, primers are used which hybridize either only to a sequencethat forms by the bisulfite treatment of a DNA which is unmethylated atthe respective position, or, vice versa, primers which bind only to anucleic acid which forms by the bisulfite treatment of a DNAunmethylated at the respective position. Amplified products can beproduced accordingly with these primers, the detection of which in turnsupplies indications of the presence of a methylated or unmethylatedposition in the sample to which the primers bind.

A newer method is also the detection of cytosine methylation by means ofa Taqman PCR, which has become known as “methyl light” (WO 00/70090). Itis possible with this method to detect the methylation state ofindividual positions or a few positions directly in the course of thePCR, so that a subsequent analysis of the products becomes superfluous.

An overview of the state of the art in oligomer array production can bederived also from a special issue of Nature Genetics which appeared inJanuary 1999 (Nature Genetics Supplement, Volume 21, January 1999), theliterature cited therein and U.S. Pat. No. 5,994,065 on methods for theproduction of solid supports for target molecules such asoligonucleotides in the case of reduced nonspecific background signal.

Probes with multiple fluorescent labels are used for scanning animmobilized DNA array. Particularly suitable for fluorescent labels isthe simple introduction of Cy3 and Cy5 dyes at the 5′-OH of therespective probe. The fluorescence of the hybridized probes is detected,for example, by means of a confocal microscope. The dyes Cy3 and Cy5,among many others, are commercially available.

Matrix-assisted laser desorptions/ionization mass spectrometry(MALDI-TOF) is a very powerful development for the analysis ofbiomolecules (Karas M, Hillenkamp F. Laser desorption ionization ofproteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988Oct. 15; 60(20):2299-301). An analyte is embedded in a light-absorbingmatrix. The matrix is vaporized by a short laser pulse and the analytemolecule is transported unfragmented into the gaseous phase. The analyteis ionized by collisions with matrix molecules. An applied voltageaccelerates the ions in a field-free flight tube. Ions are acceleratedto varying degrees based on their different masses. Smaller ions reachthe detector sooner than large ions.

MALDI-TOF spectroscopy is excellently suitable for the analysis ofpeptides and proteins. The analysis of nucleic acids is somewhat moredifficult (Gut, I. G. and Beck, S. (1995), DNA and Matrix Assisted LaserDesorption Ionization Mass Spectrometry. Molecular Biology: CurrentInnovations and Future Trends 1: 147-157). For nucleic acids, thesensitivity is approximately 100 times poorer than for peptides anddecreases overproportionally with increasing fragment size. For nucleidacids, which have a multiply negatively charged backbone, the ionizationprocess via the matrix is essentially less efficient. In MALDI-TOFspectroscopy, the choice of matrix plays an eminently important role.Several very powerful matrices, which produce a very finecrystallization, have been found for the desorption of peptides. In themeantime, several effective matrices have been developed for DNA, butthe difference in sensitivity was not reduced thereby. The difference insensitivity can be reduced by modifying the DNA chemically in such a waythat it resembles a peptide. Phosphorothioate nucleic acids, in whichthe usual phosphates of the backbone are substituted by thiophosphates,can be converted by simple alkylation chemistry into a charge-neutralDNA (Gut, I. G. and Beck, S. (1995), A procedure for selective DNAalkylation and detection by mass spectrometry. Nucleic Acids Res. 23:1367-1373). The coupling of a “charge tag” to this modified DNA resultsin an increase in sensitivity by the same amount as is found forpeptides. Another advantage of “charge tagging” is the increasedstability of the analysis in the presence of impurities, which make thedetection of unmodified substrates very difficult.

Genomic DNA is obtained from DNA of cells, tissue or other test samplesby standard methods. This standard methodology is found in referencessuch as Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual,1989.

After PCR was invented, numerous variants became known in the next fewyears, which refine this technique for the amplification of DNA. Inparticular, multiplexing of the PCR (multiplex PCR) should be mentionedhere, in which more than 2 specific primers are used and thus aplurality of different, specific amplifications can be produced in onereaction vessel. Particularly interesting also is so-called nested PCR,which is used, among other things, for the detection of particularlysmall DNA quantities. This type of PCR is comprised of twoamplifications, one following the other, wherein the primers of thesecond amplification lie within the first amplified product and are notidentical with the primers of the first amplification. In this way, aparticular specificity is achieved, since the primers of the secondamplification only function if the intended fragment was produced in thefirst amplification. In contrast, the propagation of any possiblebyproducts of the first amplification in the second amplification isexcluded as much as possible.

The present methods for methylation analysis, which contain a bisulfitereaction, without exception, have the disadvantage that the reactionsolution cannot be utilized directly for a subsequent polymerase chainreaction, since the high salt content of the bisulfite reaction acts ina disruptive manner. Thus, in practice, several purification and/orwashing steps must be conducted, which contribute, particularly in thecase of small quantities of DNA sample, to the poor reproducibility ofthe protocols, the troublesome handling and the low sensitivity of themethods. Also, the DNA must first be isolated before it can be utilizedin the bisulfite reaction, as is also the case for other molecularbiological assays.

The object of the present invention is thus to overcome thedisadvantages of the prior art.

The object is solved by a method for the analysis of cytosinemethylation patterns in genomic DNA samples, whereby the followingmethod steps are conducted:

a) the genomic DNA is isolated from cells or other accompanyingmaterials and bound essentially irreversibly to a surface;b) the DNA bound to the surface is treated, preferably with a bisulfite(=disulfite, hydrogen sulfite), in such a way that cytosine is convertedinto a base that is different in its base pairing behavior in the DNAduplex, while 5-methylcytosine remains unchanged;c) the reagents used in step b) are removed in a washing step;d) selected segments of the immobilized DNA are amplified in apolymerase reaction;e) the amplified products are investigated with respect to theirsequence.

It is advantageous if the following additional steps are conducted:

f) the reagents and products of the polymerase reaction are removed in awashing step;g) other selected segments of the immobilized DNA, which are differentfrom those in step d), are amplified in a polymerase reaction;h) the amplified products are investigated with respect to theirsequence.

In addition, it is particularly advantageous according to the inventionthat steps f-g) are repeated several times, whereby in eachamplification according to step g), segments other than those in one ofthe preceding amplifications are amplified.

It is preferred according to the invention that the binding of the DNAto the surface is covalent.

It is particularly advantageous that the DNA is also isolated directlyin the immobilization step.

It is preferred according to the invention that the DNA is isolated fromwhole blood or blood serum. It is also advantageous according to theinvention that the DNA is isolated from lysed tissue.

It is thus preferred according to the invention that the lysis isconducted by means of proteinase K.

It is particularly preferred according to the invention thatimmobilization is conducted in the wells of a microtiter plate with 96wells or 384 wells, whereby different DNA samples are immobilized in thewells.

It is particularly advantageous according to the invention that theimmobilization is conducted in PCR reaction wells, whereby different DNAsamples are immobilized in the wells. It is preferred according to theinvention that the DNA is immobilized on a metal oxide, preferablyaluminum oxide.

The method according to the invention is also advantageous ifimmobilization is made on a hydrophobic material and the binding isessentially irreversible only under selected buffer conditions.

It is also preferred according to the invention that an amplificationstep is conducted with several pairs of primers as a multiplex PCR.

It is thus preferred according to the invention that all amplifiedproducts of an immobilized DNA sample, or a large portion thereof, arepooled, and thus are jointly introduced to further analysis. A largeportion is approximately 50% or more of the amplified products. However,it can also be up to 75% or more.

It is also preferred in the method according to the invention that thisfurther analysis involves the hybridization to an oligonucleotide arrayor PNA (peptide nucleic acid) array.

It is also preferred according to the invention that the analysis isconducted during the amplification by means of a realtime PCR method.

It is also advantageous according to the invention that the analysis isconducted after the amplification in the same reaction well by plottinga melting-point curve.

It is particularly preferred according to the invention that theanalysis is conducted by allele-specific hybridization ofoligonucleotides or PNAs (peptide nucleic acids) at the positions to beinvestigated in the amplified products.

It is further preferred according to the invention that the analysis isconducted by hybridization of oligonucleotide primers and a subsequentprimer extension reaction or a sequencing reaction.

The subject of the present invention is also the use of the methodaccording to the invention for the diagnosis and/or prognosis of adverseevents for patients or individuals, whereby these adverse events belongto at least one of the following categories: undesired druginteractions; cancer diseases; CNS malfunctions, damage or disease;symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damage; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease, malfunction and damage; malfunction,damage or disease of the gastrointestinal tract; malfunction, damage ordisease of the respiratory system; lesion, inflammation, infection,immunity and/or convalescence; malfunction, damage or disease of thebody as a consequence of an abnormality in the development process;malfunction, damage or disorder of the skin, the muscles, the connectivetissue or the bones; endocrine and metabolic malfunction, damage ordisease; headaches or sexual malfunction.

The use of a method according to the invention is preferred according tothe invention for distinguishing cell types or tissues or forinvestigating cell differentiation.

The subject of the present invention is also a kit, comprised of areagent for the treatment of DNA according to step b, at least twoprimer oligonucleotides for producing the amplified products, a solidphase for immobilizing the sample DNA, as well as, optionally, othersolutions, and instructions for conducting an assay according to amethod according to the invention.

The solution to the problem which is the basis for the present inventionconsists of the fact that the DNA, which is bound to a solid phase inthe scope of its aimed-at isolation from, for example, whole blood,blood serum or tissue, is also directly subjected to a subsequentbisulfite reaction on this solid phase, without anything further. Afterthe very simple removal of the bisulfite reaction mixture, which can beachieved in this case by post-washing with water or a suitable buffer,the immobilized DNA can be utilized directly also for the amplification.Alternatively, it can be stored in this immobilized form and used foramplification only as needed. Since the immobilized DNA is notsubstantially changed by the amplification, it is also possible toconduct several subsequent amplifications with the immobilized DNA as atemplate, after the reaction components of each preceding amplificationhave been removed by washing steps.

Overall, the present invention thus provides a method, which representsa considerable simplification with respect to conducting any methylationassay based on bisulfite treatment. The sample DNA need only be bounddirectly to a solid phase; the bisulfite treatment will be conducted onthis solid phase and subsequently a polymerase reaction will beconducted also with the use of the same solid phase. This also permitsconducting stable assays starting from very small DNA quantities, suchas those from blood serum.

In a meaningful way, the solid phase is a modified surface of a well, inwhich the PCR reaction is then also conducted, and advantageously, acommercially available PCR well, which may also be present as a figure-8strip or as part of a microtiter plate. The essential object of thepresent invention was thus to provide surfaces, among other things,which, first of all, can bind the DNA irreversibly as much as possible,and secondly, however, also remain sufficiently stable under theconditions prevailing in the bisulfite treatment and additionally keepthe DNA bound.

Two surfaces were identified which fulfill this objective. The first isaluminum oxide, and the second, C18-alkyl chains, which permit solidbinding of the DNA in combination with suitable cations, such astriethylammonium ions. C18-alkyl chains can be introduced by means of anoctadecyl trialkoxysilane by the silanizing method, which is known bythe person of average skill in the art. Methods for modifying surfaceswith aluminum oxide are described in U.S. Pat. No. 6,291,166, amongother [publications].

The method according to the invention for the analysis of cytosinemethylation patterns is comprised of the following substeps:

1. The genomic DNA is isolated from cells or other accompanyingmaterials and bound essentially irreversibly to a surface.2. The DNA bound to the surface is treated, preferably with a bisulfite(=disulfite, hydrogen sulfite), in such a way that cytosine is convertedinto a base that is different in its base pairing behavior in the DNAduplex, while 5-methylcytosine remains unchanged.3. The reagents used in the second step are removed in a washing step.4. Selected segments of the immobilized DNA are amplified in apolymerase reaction and5. The amplified products are investigated with respect to theirsequence.

In a particularly preferred variant of the method, the following stepsare additionally conducted:

6. The reagents and products of the polymerase reaction are removed in awashing step.7. Other selected segments of the immobilized DNA, which are differentfrom those in step d), are amplified in a polymerase reaction.8. The amplified products are investigated with respect to theirsequence.

In another particularly preferred variant of the method, steps 6-8 arerepeated several times.

In the first method step, the preferably genomic DNA is isolated fromcells or other accompanying materials and bound essentially irreversiblyto a surface.

An irreversible binding in the sense of the present invention means abinding, which cannot be completely separated again with the meansusually available under the conditions prevailing in the reaction. Thisbinding may preferably involve a covalent binding, an ion-pair binding,or, however, a binding which is based on electrostatic or hydrophobiceffects.

The DNA is preferably isolated in such a way that a body fluid or,however, a lysate of a tissue is contacted with the surface, which inturn preferably binds the DNA irreversibly. A special buffer (forexample, triethylammonium acetate) is required for this purpose in thecase of the C18 material. The supernatant is removed and it ispost-washed either with buffer or water (or both), in order to conductthe subsequent bisulfite reaction with initial material that is aspurified as possible.

In a particularly preferred variant of the method, the binding of theDNA to the surface is covalent. In another particularly preferred methodvariant, the DNA is also isolated directly in the immobilization step.The DNA is preferably isolated from whole blood or blood serum.

In another particularly preferred method variant, the DNA is isolatedfrom lysed tissue. The lysis is particularly preferably conducted bymeans of proteinase K.

In another particularly preferred method variant, immobilization isconducted in the wells of a microtiter plate with 96 wells or 384 wells,in which different DNA samples are immobilized in the wells.

In a particularly preferred method variant, the immobilization isconducted in PCR reaction wells, whereby different DNA samples areimmobilized in the wells.

The DNA is particularly preferably immobilized to a metal oxide,preferably aluminum oxide. In another particularly preferred methodvariant, the immobilization is made on a hydrophobic material and thebinding is essentially irreversible only under selected bufferconditions.

The DNA to be analyzed is obtained preferably from the usual sources forDNA, such as, e.g., cell lines, blood, sputum, stool, urine,cerebrospinal fluid, tissue embedded in paraffin, for example, tissuefrom eyes, intestine, kidney, brain, heart, prostate, lung, breast orliver, histological slides and all other possible combinations thereof.

In the second step of the method, the DNA bound to the surface ispreferably treated with bisulfite (=disulfite, hydrogen sulfite) in sucha way that all of the cytosines not methylated at the 5-position of thebase are modified such that a base that is different with respect to itsbase pairing behavior is formed, whereas the cytosines that aremethylated in the 5-position remain unchanged.

If a bisulfite reagent is used, trialkylammonium bisulfite isparticularly preferred in the case of the C18 surface, in order toassure a solid binding of the DNA to the surface. In the case of thealuminum oxide surface, sodium bisulfite is preferably used.

The DNA sample is particularly preferably denatured prior to thetreatment, either thermally or with the use of an alkaline reagent, suchas, for example, dilute (preferably 0.1 to 0.3 M) sodium hydroxide.

If bisulfite is used for the reaction, then an addition occurs on theunmethylated cytosine bases. For the method according to the invention,a denaturing reagent or solvent as well as a radical trap are alsopreferably present.

The following compounds or compound classes are preferably considered asthe denaturing reagents or solvents: polyethylene glycol dialkyl ethers,dioxane and substituted derivatives, urea or derivatives, acetonitrile,primary alcohols, secondary alcohols, tertiary alcohols, diethyleneglycol dialkyl ethers, triethylene glycol dialkyl ethers, tetraethyleneglycol dialkyl ethers, pentaethylene glycol dialkyl ethers, hexaethyleneglycol dialkyl ethers, DMSO or THF. Of course, the DNA may also beembedded in agarose after the denaturing, by adding the agarose indissolved form and subsequent cooling, analogous to the method publishedby Olek et al. The agarose can be removed thermally after the bisulfitetreatment with hot buffer or hot water.

The subsequent alkaline hydrolysis (preferably: Tris buffer pH 10 orammonia) then leads to the conversion of unmethylated cytosinenucleobases to uracil. After this, the desulfonation of the DNA (10-30min, 90-100° C.) at alkaline pH is then preferably conducted.

In the third step of the method, the previously used reagents areremoved in a washing step. It is again important that the immobilized,now chemically treated DNA that is present remains bound to the surface.This is simple in the case of a covalent binding, for example, at thesurface. In contrast, an appropriate buffer is necessary, if binding hasbeen produced, for example, via triethylammonium cations to a C18 phase.The latter also requires a buffer which promotes the binding of the DNAto the hydrophobic phase, such as, for example, a triethylammoniumacetate buffer, in the washing steps.

Preferably, several washing steps are conducted, which are particularlypreferably comprised of an automated pipetting step, in which water orbuffer is added, and a subsequent pipetting step, in which therespective buffer or the water is again removed. This can be done, forexample, by an immobilization of the DNA in a microtiter plate and withthe use of a commercially available pipetting robot (e.g., made by thecompanies Tecan or Qiagen).

In the fourth step of the method, selected segments of the immobilized,treated DNA are amplified.

The DNA sample is amplified in a polymerase chain reaction, preferablywith a heat-stable DNA polymerase. The amplification of several DNAsegments is preferably conducted in one reaction vessel or well.

The method step also can preferably be carried out in two substeps. Onebegins with a PCR pre-amplification with at least one pair of primers ofdifferent sequence, which nonspecifically hybridize the pretreated DNAsample and thus more than one amplified product results in the PCR step.Then a PCR amplification of the product formed in the pre-amplificationis conducted with primers of different sequence, which are eachidentical or inversely complementary to a segment of the pretreated DNAsample [(+) strand or (−) strand], and which hybridize specifically tothe DNA to be amplified.

In a particularly preferred variant of the method, an amplification stepis conducted with several pairs of primers as a multiplex PCR. It isalso particularly preferred that all amplified products of animmobilized DNA sample, or a large portion thereof, are pooled, and thusare jointly introduced to further analysis. After the amplification, itis particularly preferred to remove the reaction mixture from thereaction vessel or well, to which the immobilized DNA is bound. Thus theimmobilized DNA is made available as a template for furtheramplifications, preferably again with primers that have not been usedpreviously.

In a particularly preferred method variant, the amplification is thusrepeated several times with different primers, so that the method hasthe following additional steps:

1) the reagents and products of the polymerase reaction are removed in awashing step;2) other selected segments of the immobilized DNA, which are differentfrom those that were previously amplified, are now amplified in apolymerase reaction;3) the amplified products are investigated with respect to theirsequence.

In a particularly preferred variant of the method, these steps arerepeated several times, whereby in each amplification according to step2), segments other than those in one of the preceding amplifications areamplified.

In the last step of the method, and even if the above additional stepsare conducted, each of the amplified products is investigated withrespect to its sequence. The methylation state of selected cytosinebases in the DNA sample can be directly determined in this way.

This sequence analysis and the subsequent determinations of methylationstate can be produced in principle with the use of many methods, whichare also described in the prior art and are known by the person ofaverage skill in the art.

Analysis by hybridization of the amplified products on anoligonucleotide array or PNA (peptide nucleic acid) array isparticularly preferred

It is also preferred that the analysis is conducted during theamplification by means of a realtime PCR method. A variant of theinvention in which the extraction of the DNA, the bisulfite treatment,the amplification and the detection, which is preferably performed bymeans of realtime PCR, thus all of the steps, can be conducted in onereaction vessel or well is particularly preferred. In this connection, amethod is also particularly preferred, in which the analysis isconducted after the amplification in the same reaction vessel by theplotting of a melting-point curve and the base composition of thefragment and thus the methylation state can be determined from themelting behavior.

The analysis can also be conducted by introducing the surface into amass spectrometer, which determines the molecular mass of the amplifiedproducts, of fragments of the amplified products, or, however, ofprobes, which specifically hybridize to the amplified products. Thisinformation can be drawn on in turn for identifying sequences if thesequence is already known for the most part. It is also possible tointroduce the dissolved amplified products separately into a massspectrometer and to conduct the analysis according to methods known tothe person of average skill in the art.

A method is also particularly preferred, in which the analysis isconducted by allele-specific hybridization of oligonucleotides or PNAs(peptide nucleic acids) at the positions to be investigated in theamplified products.

In another particularly preferred variant of the method, the analysis isconducted by hybridization of oligonucleotide primers and a subsequentprimer extension reaction or a sequencing reaction.

The subject of the present invention is also the use of theabove-described method for the diagnosis and/or prognosis of adverseevents for patients or individuals, whereby these adverse events belongto at least one of the following categories: undesired druginteractions; cancer diseases; CNS malfunctions, damage or disease;symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damage; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease, malfunction and damage; malfunction,damage or disease of the gastrointestinal tract; malfunction, damage ordisease of the respiratory system; lesion, inflammation, infection,immunity and/or convalescence; malfunction, damage or disease of thebody as a consequence of an abnormality in the development process;malfunction, damage or disorder of the skin, the muscles, the connectivetissue or the bones; endocrine and metabolic malfunction, damage ordisease; headaches or sexual malfunction.

The use of a method is also preferred for distinguishing cell types ortissues or for investigating cell differentiation.

The subject of the present invention is also a kit, comprised of areagent for the treatment of DNA, at least two primer oligonucleotidesfor producing the amplified products, a solid phase for immobilizing thesample DNA, as well as, optionally, other solutions, and instructionsfor conducting at least one of the above-described method variants.

EXAMPLE Bisulfite Treatment of Promega DNA and M13 DNA in DerivatizedReaction Wells Binding of the DNA

Genomic DNA (Promega) cleaved by EcoRI and M13 plasmid DNA were used forthe binding of DNA to the surface of reaction wells coated with aluminumoxide. 160 ng were pipetted each time into the corresponding reactionwells, which were then filled with water to a total volume of 20 μl,mixed briefly on a shaker and incubated for 15 minutes at roomtemperature. Then the solution was removed and the wells were washedtwice, each time with 50 μl of water. In order to reduce the activity ofthe remaining binding sites on the tube surface, 10 μl of a 5% bovineserum albumin solution were pipetted in, then the well was filled with40 μl of water and incubation was conducted at room temperature for 15minutes. Then the wells were washed once with 50 μl of water each.

Bisulfite Treatment

The bound DNA was denatured at 96° C. without addition of water for 20minutes in an Eppendorf Mastercycler. The wells were then removed asquickly as possible and mixed with 6 μl of dioxane, whereby the DNAremained denatured. For the bisulfite reaction, 10 μl of a 0.75 M sodiumbisulfite solution, 2 μl of a radical trap(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 1ml of dioxane) und 2 μl of water were added. The reaction wells wereincubated at 50° C. in an Eppendorf Mastercycler for five hours.

Desulfonation

After the bisulfite reaction was carried out, the solutions werepipetted out and the wells were washed with 100 μl of water and, inpreparation for the desulfonation, with 100 μl of a 50 mM Tris-HClsolution. The desulfonation was carried out with 50 μl of a 50 mMTris-HCl solution at pH 9 for 20 minutes at 96° C. After 3× washing with50 μl of water each time, the reaction wells were ready for anamplification by means of PCR.

PCR

The PCR was conducted on a scale of 25 μl. The following served asprimers for the Promega DNA: 5′-TAA GTA TGT TGA AGA AAG ATT ATT GTA G-3′and 5′-TAA AAA CTA TCC CAT AAT AAC TCC CAA C-3′, and the followingprimers were used for the M13 plasmid DNA: 5′-ATT ACA AAA TCG CGC AAA-3′and 5′-AAG TCG GAG GTT AAA AAG GT-3′ (MWG). The two primers were placedeach time in a solution with a concentration of 12.5 pmol/μl and 2 μl ofthis solution of primer pairs were pipetted into the corresponding tube.For the PCR, 2.5 μl of dNTP Mix (Fermentas, concentration of each dNTP:2.5 μmol/μl), 0.3 μl Hot Star Taq (Qiagen), 2.5 μl 10×PCR BufferSolution (Qiagen, 15 mmoles of MgCl₂ contained in the buffer) and 17.7μl of water (Fluka) were placed in the wells for each batch.

The PCR was monitored by gel electrophoresis. For this purpose, 5 ul ofthe sample with 3 μl of loading dye were applied onto a 1.4% agarose gel(Eurogentec, Inc.), and 1×TBE served as the running buffer. Thefragments were stained with ethidium bromide and the gel wasphotographed in UV.

1. A method for the analysis of cytosine methylation patterns in genomicDNA samples, said method comprising the steps of: a) isolating thegenomic DNA from cells or other accompanying materials and immobilizingthe isolated genomic DNA to a surface by covalent bonding; b) treatingthe immobilized DNA in such a way that cytosine is converted into a basethat is different in its base pairing behavior in the DNA duplex, while5-methylcytosine remains unchanged; c) removing the reagents used instep b) in a washing step; d) amplifying selected segments of theimmobilized DNA in a polymerase reaction; e) investigating the amplifiedproducts with respect to their sequence.
 2. The method according toclaim 1, further comprising the steps of: f) removing the reagents andproducts of the polymerase reaction in a washing step; g) amplifyingother selected segments of the immobilized DNA, which are different fromthose in step d), in a polymerase reaction; h) investigating theamplified products with respect to their sequence.
 3. The methodaccording to claim 2, further comprising repeating steps f)-g) severaltimes, whereby in each amplification according to step g), segmentsother than those in one of the preceding amplifications are amplified.4. The method according to claim 1, wherein the treating step comprisestreating the immobilized DNA with a bisulfite.
 5. The method accordingto claim 1, wherein the DNA is also isolated directly in theimmobilizing step.
 6. The method according to claim 5, wherein the DNAis isolated from whole blood or blood serum.
 7. The method according toclaim 5, wherein the DNA is isolated from lysed tissue.
 8. The methodaccording to claim 7, wherein the lysis is conducted by means ofproteinase K.
 9. The method according to claim 1, wherein theimmobilization is conducted in the wells of a microtiter plate with 96wells or 384 wells, in which different DNA samples are immobilized inthe wells.
 10. The method according to claim 1, wherein theimmobilization is conducted in PCR reaction wells, whereby different DNAsamples are immobilized in the wells.
 11. The method according to claim1, wherein the isolating of the DNA occurs on a metal oxide.
 12. Themethod according to claim 1, wherein the immobilization is made on anon-hydrophobic surface.
 13. The method according to claim 1, whereinthe amplification step is conducted with several pairs of primers as amultiplex PCR.
 14. The method according to claim 1, wherein allamplified products of an immobilized DNA sample are pooled, and thus arejointly introduced to further analysis.
 15. The method according toclaim 14 wherein said further analysis involves the hybridization to anoligonucleotide array or PNA (peptide nucleic acid) array.
 16. Themethod according to claim 1, wherein the investigating step is conductedduring the amplification by a realtime PCR method.
 17. The methodaccording to claim 1, wherein the investigating step is conducted afterthe amplification in the same reaction well and comprises plotting amelting-point curve.
 18. The method according to claim 1, wherein theinvestigating step comprises allele-specific hybridization ofoligonucleotides or PNAs (peptide nucleic acids) at the positions to beinvestigated in the amplified products.
 19. The method according toclaim 1, wherein the investigating step comprises hybridization ofoligonucleotide primers and a subsequent primer extension reaction or asequencing reaction.
 20. A method for the conversion of unmethylatedcytosine in genomic DNA samples, said method comprising the steps of: a)isolating the genomic DNA from cells or other accompanying materials andimmobilizing the isolated genomic DNA to a surface by covalent bonding;b) treating the immobilized DNA with a bisulfite in such a way thatcytosine is converted into a base that is different in its base pairingbehavior in the DNA duplex, while 5-methylcytosine remains unchanged;and c) removing the reagents used in step b) in a washing step.
 21. Akit, comprised of a reagent for the treatment of DNA according to claim1, at least two primer oligonucleotides for producing the amplifiedproducts, a solid phase for immobilizing the sample DNA, as well as,optionally, other solutions, and instructions for conducting an assayaccording to claim 1.